The oil palm is one of the world’s most economically important oil-producing crops and is a major source of vegetable oil in tropical regions (Soonsuwonet et al.,  2020). Its economic importance is largely attributed to its high productivity and wide-ranging uses in the food, cosmetics, oil chemistry and biofuel industries. It has long been recognized as a key plantation crop, particularly in Southeast Asia, which represents the principal center of its cultivation and commercial production (Abubakar et al., 2023). Owing to its diverse industrial applications and substantial economic value, oil palm has become the most widely traded vegetable oil crop worldwide (Sparjanbabu et al., 2019). The coconut is another tropical crop of great economic importance, widely cultivated in coastal regions of Asia, Africa and the Pacific Islands. It plays a vital role in the livelihoods of millions of people and is a major source of food, oil, fiber and industrial raw materials. Like oil palms, coconuts contribute significantly to the global supply of vegetable oils and support various agro-industrial sectors (Henrietta et al.,  2022). However, the fungus Marasmiellus palmivorus has been reported as an important pathogen affecting both coconut and oil palms, as it is associated with the formation of fruiting bodies and disease development in infected plant tissues.

Marasmiellus is a genus of fungi belonging to the family Marasmiaceae. This widely distriduted genus was first circumscribed by American mycologist William Murrill in 1915 (Antonín and Noordeloos, 1993; Corner, 1996). The genus comprises approximately 400 species and is distributed throughout most regions of the world (Takahashi and Degawa, 2006). Several species of Marasmiellus are plant pathogenic fungi with an extensive host range that cause damage to a wide range of plants, including economically important crops, such as sugar cane, corn, beech grass, banana, orchid, maize, cacao, small cardamom and palm (Dhanya et al., 2021; Amoako-Attah et al., 2025). In Iraq, M. palmivorus was reported as the causal agent of reed wilt disease (Tamur et al., 2018). The fungus’s growth behavior and habitat suggest it is likely to occur in all countries where oil palm and coconut are cultivated commercially. The fungus attacks the fruit and the petiole of the oil palm (Maizatul-Suriza et al., 2021). Furthermore, M. palmivorus caused Marasmeillus embryo and shoot rot (MESR), a common disease of coconut seed nuts in the germination bed, which attacks and kills the embryo or the developing shoot (Almaliky et al., 2013).

There is a lack of information available on the characterization and pathogenicity of this species in Malaysia. A regular supply of basidiocarps is necessary for the identification of the species in the absence of identifiable sexual fruiting bodies or asexual spores (basidiocarps or basidiospores), investigation of the processes of pathogenesis and to study the characterization to aid research programs and to provide information that might lead to a better understanding of the behavior of this species of fungus in the field. However, there were successful attempts to develop several methods for the production of basidiocarps on a closely related pathogen (also Marasmiaceae), Crinipellis perniciosa (Stahel) Singer, the causal agent of witches’ broom disease of cocoa (Theobroma cacao L.). Purdy and Dickstein (1990) have reproduced the basidiocarp artificially in 4-5 weeks by growing sterilized mycelial mats, hung on sterile cocoa brooms and simulating tropical conditions. Given the importance of basidiocarp production for research on pathogenic agaric fungi, this study aimed to establish alternative techniques for basidiocarp production in Marasmiellus palmivorus, a significant pathogen of oil palm and coconut, using natural culture media in a glasshouse environment. These methods are expected to be valuable for researchers in the field of plant pathology.

Isolation and perpetuation of Marasmiellus palmivorus cultures
 
Marasmiellus palmivorus isolates were recovered from symptomatic coconut seed nuts, oil palm fruits, basidiocarps and rhizomorphs collected from Selangor (Bangi, Malaysian Palm Oil Board (MPOB) and University Putra Malaysia (UPM)) and Perak (United Plantation Berhad), Malaysia. Diseased tissues and basidiocarps were surface-sterilized with 1.25% NaOCl for 1 min, rinsed, sectioned (»1 cm²) and plated on 1.5% water agar supplemented with 85% lactic acid (1 ml L^-1). Isolates from rhizomorph segments were directly plated on the same medium. Pure cultures were transferred to Malt Extract Agar (MEA) and colony morphology was recorded. Isolates were identified using macroscopic and microscopic characteristics following standard procedures (Turner, 1981). If basidiocarps and basidiospores could not be identified, isolates were tentatively considered M. palmivorus based on disease symptoms and the presence of clamp connections at septa. The diameter of 20 mature basidiocarps and the length of their stipes were measured, as well as the length, breadth and length/breadth ratio of 100 basidiospores from basidiocarps. All isolates were preserved on MEA in slant tubes at 4°C in the Plant Pathology Laboratory at University Putra Malaysia (UPM) until use.
 
Pathogenicity test of Marasmillus palmivorus
 
To confirm the pathogenicity of Marasmiellus palmivorus, isolates obtained from symptomatic tissues were inoculated onto healthy oil palm and coconut seedlings. Each isolate was cultured in 100 mL malt extract broth in 250 mL Erlenmeyer flasks and incubated at 27±2°C for five days on an orbital shaker at 125 rpm. The cultures were filtered through sterile cheesecloth and the harvested mycelia were homogenized in 100 mL sterile distilled water to prepare the inoculum. Small wounds were made at the basal stem and root regions of each seedling using a sterile scalpel, followed by inoculation with 10 ml of mycelial suspension. Control plants received only sterile distilled water. The inoculated seedlings were transplanted into pots containing sterilized sand, soil and peat moss (3:2:1, v/v/v) and maintained in a glasshouse at 29±4°C and 85-95% relative humidity. Pathogenicity was confirmed by the development of symptoms similar to those observed under natural infection and by re-isolation of the pathogen from infected tissues.
 
Fruiting body (basidiocarps) production by the wheat grain method
 
The spawn was prepared using 100 g of wheat grains (Triticum aestivum), which were washed under running tap water, placed in polypropylene bags and soaked in distilled water for 24 h (15 x 30 cm). The bags were then autoclaved at 121°C for 20 min. After cooling, the wheat grain medium was inoculated with five mycelial plugs (5 mm diameter) taken from the actively growing margins of 5-day-old cultures grown on Malt Extract Agar (MEA). The inoculated bags were incubated in darkness at room temperature (28±2°C) for two weeks and shaken every other day to ensure uniform mycelial colonization. The potting substrate consisted of soil, peat and sand mixed at a ratio of 3:2:1 (v/v/v). To avoid contamination of the soil, the described soil mixture was steam-treated at 100°C for 1.5 h. Once the mycelium had covered the surface of the wheat grain media, the polypropylene bag was removed and the colonized substrate was embedded into 25-cm-diameter pots containing the prepared substrate and covered with a thin layer of the same soil mixture as a casing layer. The pots were watered and covered with a polyethylene bag for 4 days to provide the fungus with favorable conditions for growth (Fig 1A). They were placed on a bench in the glasshouse and kept moist by a sprinkler.


 
Fruiting body (basidiocarps) production by the rubber wood method
 
Fresh rubber wood (Hevea brasiliensis) blocks (5 x 5 x 2 cm) were prepared from mature rubber trees. The blocks were placed in polypropylene bags and soaked in distilled water for 24 hours. They were autoclaved at 121°C for 1 hour and cooled before adding 50 ml of malt extract agar (MEA) and autoclaved again at 121°C for 20 min. The wood blocks were rotated to ensure uniform agar coating before solidification. Once cooled, the blocks were inoculated with five mycelial plugs (5 mm in diameter) of either isolate. The plugs were obtained from the growing margins of 5-day-old cultures on MEA. The bags were incubated in darkness at room temperature (28±2°C) for four weeks. After the mycelium covered the block surfaces, the polypropylene bag was removed and the colonized blocks were embedded on the surface of the soil in the 25-cm-diameter pots filled with the prepared soil mixture. The pots were watered and covered with a polyethylene plastic bag for four days (Fig 1B). They were placed on a bench in the glasshouse and kept moist by a sprinkler. Two separate experiments were performed for five isolates from oil palm and five isolates from coconut, with four replicates per treatment per experiment. Therefore, greenhouse conditions were set at natural light cycles of 12 h: 12 h (day: night), 29±4°C and a high relative humidity level (85-95%). Basidiocarps of M. palmivorus were counted weekly for 10 weeks. The diameter of 20 pilei and the length of stipes were measured; in addition, the lengths, breadth and length/breadth of 100 basidiospores from three basidiocarps in each treatment were also measured. The ability of basidiospores to germinate was tested by allowing basidiospores to deposit from the basidiocarp on the surface of a sterile Petri dish. These spores were suspended in sterile distilled water. A drop of spore suspension was placed in each well of a cavity slide containing a few drops of sterile distilled water. The slides were incubated in the dark at a moist chamber under laboratory conditions (28±2°C). A drop of clear lacto phenol was added to each cavity slide before being observed under a light microscope. Spore germination was assessed at 6, 12 and 24 h after incubation. A minimum of 100 basidiospores was assessed in each of four replications of cavity slides. The percentage of germination was recorded.
 
Statistical analysis
 
The pots were set up in a completely randomized design. The data obtained were analyzed using analysis of variance (ANOVA) via the General Linear Model (GLM) procedure in SAS. Means separation by LSD test (P=0.05) was carried out for comparison of the total number of basidiocarps produced.

A total of ten different isolates of Marasmiellus palmivorus were isolated during this study: 5 isolates, UPM4, Bangi1, MPOB3, OP2 and OP4 from oil palm and 5 isolates, C1, C2, C3, C5 and C6 from coconut seed nuts (Table 1). All isolates showed clamp connections under the light microscope. The results confirm that all isolates were pathogenic and showed disease symptoms on the oil palm and coconut seedlings. All the different isolates of M. palmivorus successfully produced high numbers of basidiocarps by these two methods (Fig 2 and 3). When the wheat grain method was used, basidiocarps of M. palmivorus were produced after 2 weeks’ embedment onto a sterilized soil in the glasshouse, while it takes 4 weeks in the rubber wood method.







The results obtained for the total number of basidiocarps produced from the oil palm and coconut isolates were presented in Table 2 and 3, respectively. The two different methods affected basidiocarp production markedly. Comparisons between the two methods showed that the wheat grain technique resulted in a much higher production of basidiocarps compared to the rubber wood method for all isolates of M. palmivorus. In oil palm isolates, as the results showed in Table 2, the total number of basidiocarps produced by the isolates Bangi1 and OP4 (188,185), respectively, was significantly greater than in the rubber wood method (40, 37), respectively. Also, there was no significant difference between these two isolates. The total number of basidiocarps produced from each of the three isolates, UPM4, MPOB3 and OP2, was significantly different in both methods (117, 96 and 78) and (23, 16 and 12), respectively. The results in Table 3 showed that the isolates C6 and C3 from coconut produced a higher number of basidiocarps (184 and 180) in the wheat grain method and they were significantly different from the other isolates C5, C1 and C2 (83,82 and 80), respectively. However, there were no significant differences among these three isolates. In the rubber wood method, all of the 5 isolates of M. palmivorus produced a lower number of basidiocarps than the wheat grain method and there were no significant differences between C6 and C3 (42, 41). Also, no significant difference between C5, C1 and C2 (21,17 and 16).





The results presented in Table 4 and 5 showed that the morphology and characterization of basidiocarps and basidiospores of all isolates of M. palmivorus obtained from wheat grain and rubber wood methods were comparable to those documented earlier on natural basidiocarps in the field, even the (UPM4, OP4) and (C5, C6) that were isolated from diseased samples and Rhizomorphs. In M. palmivorus, which was isolated from oil palm, the pileus is white, pale orange in the middle, with a depressed center, margin curved when young, with some grooving on the margin when mature, 9-30mm in diameter. The stipe is white, whitish, towards the base, pale orange, silky, stuffed, then becomes hollow, the base bulbous, eccentric, mostly curved, 7-29 mm [Fig 2 (A) and Fig 3(C)]. The spores were hyline, oblong to ellipsoid and smooth. In M. palmivorus that was isolated from coconut, the pileus is white-cream, pale cinnamon in the middle, with a depressed center, with some grooving on the margin when mature, 7-20 mm in diameter. The stipe is white or whitish, light cinnamon brownish in the base, solid, eccentric, mostly curved, 7-29 mm [Fig 2(B) and Fig 3(D)]. The spores were hyline, ellipsoid and smooth. The basidiospores obtained from all of the isolates by using the two methods showed high germination ability after 6h on a microscope, a cavity slide reaching 80-85%.





This research is the first account of the production of basidiocarps from mycelial Cultures of M. palmivorus in the glasshouse. All the isolates successfully produced a high number of basidiocarps on the wheat grain and rubber wood methods. Successful basidiocarp production was attributed to the right environmental conditions in the glasshouse, such as temperature, light and relative humidity. These factors are important, as they are with other basidiomycetes (Manachere, 1980; Rocha and Wheeler, 1985). Such environmental stimuli produced basidiocarps.  It is of particular interest that the wheat grain method produced the highest number of basidiocarps compared with the rubber wood method for all isolates from M. palmivorus. The minimum time between inoculation and basidiocarp production was 4 weeks, while in the rubber wood method was 8 weeks, because this suggests that the production might be influenced considerably by inducing some of the intracellular enzyme activity. Sanios et al. (2006) found that a higher level of β-1, 3-glucanase production was observed when the mushroom spawn was prepared by developing the mycelium on wheat grain-based substrates.  This enzyme might support the processes of cellular differentiation, such as those related to the growth phase and hyphae ramification. In addition, some isolates (Bangi1, OP4) and (C6, C3) produced the highest number of basidiocarps compared to others, which might be related to the mycelial growth rates because these isolates were found to be very fast-growing on malt extract agar (MEA) (Table 1). A vegetative growth phase is generally necessary so that a mycelium reaches a stage of fruiting maturity (Manachere, 1980).

Basidiocarps and basidiospores were morphologically similar to those observed in the field and the spores showed high viability since they germinated 6 h after being placed on a microscope cavity slide. The present methods offer a simpler procedure for producing basidiocarps in large numbers and normal size, which can be greatly used for the identification of the species in the absence of identifiable sexual fruiting bodies or asexual spores (basidiocarps or basidiospores). Furthermore, the methods will be very useful for the investigation of the processes of pathogenesis, also to study the characterization to aid research programs and to provide information that might lead to a better understanding of the behavior of this species of fungus in the field.

The authors declare that there are no competing interests.